Subcellular brain fractions were sonicated in .2% Triton X-100, .1 M Na-glycinate buffer, pH 9.5, to quantitatively extract lysosomal hydrolases and block the autolytic action of brain neuraminidase. Isoenzyme patterns of 6 acid hydrolases were studied by polyacrylamide gel electrophoresis at pH 8.8. In the lysosome-enriched fraction, two or more anodic isoenzymes of all 6 hydrolases were identified but little activity remained at the origin (cathode). In the synaptosomal fraction appreciable cathodic activities were present and the fastest bands were more acidic than those in the lysosomal fraction. (C14)Lysine and N- acetyl-(H3)mannosamine, precursor of N-acetylneuraminic (NANA), injected i.p. in rats, were rapidly incorporated into rat kidney lysosomes. On isoelectric focusing, predominantly acidic glycoproteins were labeled with pIs of 2.0 to 5.0 (H3)NANA turned over twice as rapidly as (C14)lysine. With the passage of time the (C14) and (H3) radioactivity profiles underwent a progressive shift from glycoprotein fractions of low pI originally labeled into previously unlabelled fractions with higher pIs. Thus, the pIs of the fractions maximally labelled with (C14) rose from 3.3 at 1.5 hr to 5.1, 6.5 and 7.1 at 4.5, 8.5 and 12.5 days, respectively, indicating that lysosomal enzymes are synthesized solely as highly acidic, NANA-rich glycoproteins. The basic isoenzymes in lysosomes are derived from the latter in the course of their biodegradation through cleavage of NANA residues by lysosomal neuraminidase. We shall continue to study the synthesis, turnover and physicochemical properties of several lysosomal enzymes in rat brain and kidney, including the effects of differentiation, senescence and certain experimental treatments.